The shortage of kidneys is limitation for an effective national renal transplant program. One solution is to use kidneys not now suitable for transplantation, i.e. those exposed to warm ischemia (WI) in the donor. This could increase renal transplants by 20 percent/year. To utilize these kidneys requires new preservation methods specific for WI kidneys. In this study we will develop methods to preserve the WI kidney. We will use a donor model (brain dead dog) to mimic the clinical situation. Specific Aim 1 will determine the relationship between brain death (BD) and transplant viability, including variables such as time of BD, preservation method, and length of preservation. Specific Aim 2 will determine if, in a renal transplant model, BD and WI cause greater injury to the kidney than WI by itself. These studies will define clearly the model necessary for the completion of this study. Specific Aim 3 will define the mechanisms of BD, WI, and preservation injury on the renal vascular (endothelial and smooth muscle cells) and parenchymal cell metabolism. Our hypothesis is that BD and WI, combined with cold preservation 2causes a Ca-dependent disruption of cell metabolism in both endothelial and parenchymal cells leading to increased reactive oxygen metabolite (ROM) generation, and cytoskeletal changes leading to loss of cell viability. We will test this hypothesis in vitro using vascular and parenchymal cell models. We will study how agents that modify Ca metabolism, ROM generation, and cytoskeleton changes affect viability and functions of vascular and parenchymal cells from kidneys exposed to BD, WI, and preservation. We propose that continuous hypothermic machine perfusion of kidneys will reverse some of the consequences of BD and WI that cause irreversible injury, better than simple cold storage. In Specific Aim 4, we will use the renal transplant model to develop an improved method to preserve the kidney from the BD donor after WI. Our method will be based upon perfusate modifications developed in Specific Aim 3 that are shown to improve preservation of the kidney in our in vitro models. Our goal will be to develop a new method of renal preservation, appropriate for not only the WI kidney, but also kidneys from hypotensive or older donors. This will increase the number of kidneys available for transplantation, and reduce the incidence of delayed graft function and primary nonfunction in both healthy and less-than-ideal donor kidneys.